A number of tumor necrosis factor receptor proteins (xe2x80x9cTNFR proteinsxe2x80x9d) have been isolated in recent years, having many potent biological effects. Aberrant activity of these proteins has been implicated in a number of disease states.
One such TNFR homologue, referred to herein as xe2x80x9cFAS Ligand Inhibitory Proteinxe2x80x9d or xe2x80x9cFLINTxe2x80x9d, binds FAS Ligand (FAS L) thereby preventing the interaction of FAS L with FAS (See U.S. Provisional Applications Ser. Nos. 60/112,577, 60/112,933, and 60/113,407, filed Dec. 17, 18 and 22, 1998, respectively, the entire teachings of which are incorporated herein by reference).
Increased activation of the FAS-FAS Ligand signal transduction pathway is implicated in a number of pathological conditions, including runaway apoptosis (Kondo et al., Nature Medicine 3(4):409-413 (1997); Galle et al., J. Exp. Med. 182:1223-1230 (1995)), and inflammatory disease resulting from neutrophil activation (Miwa et al.,. Nature Medicine 4:1287 (1998)).
xe2x80x9cRunaway apoptosisxe2x80x9d is a level of apoptosis greater than normal, or apoptosis occurring at an inappropriate time. Pathological conditions caused by runaway apoptosis include, for example, organ failure in the liver, kidneys and pancreas. Inflammatory diseases associated with excessive neutrophil activation include sepsis, ARDS, SIRS and MODS.
Compounds such as FLINT, which inhibit the binding of FAS to FASL, and LIGHT to LTxcex2R and/or TR2/HVEM receptors, can be used to treat or prevent diseases or conditions that may be associated with these binding interactions. The therapeutic utility of FLINT could be enhanced by FLINT analogs that exhibit modified pharmacological properties (e.g., enhanced potency, and/or longer in vivo half-lives, and/or greater affinity for FASL), modified pharmaceutical properties (e.g., decreased aggregation and surface adsorption, increased solubility and ease of formulation) and/or modified physical properties such as susceptibility to proteolysis.
The FLINT polypeptide undergoes proteolysis in vivo to produce at least two major peptide fragments. One of the fragments consists of residues 1 through 218 of SEQ ID NO:1 (alternatively residues 1 through 247 of SEQ ID NO.:3), termed herein xe2x80x9cFLINT metabolite;xe2x80x9d the other consists of residues 219 through 271 of SEQ ID NO:1 (alternatively residues 248 through 300 of SEQ ID NO:3). Cleavage at the 218 position in vitro can be achieved when native FLINT (SEQ ID NO:3), or mature FLINT (SEQ ID NO:1), is treated with a trypsin-like enzyme, for example, thrombin, trypsin or other serine protease. Thus it is likely that a serine protease is responsible for the in vivo proteolysis of FLINT. Production of FLINT metabolite is disclosed in co-pending U.S. patent application Ser. No. 09/936,024, herein incorporated by reference.
In vitro studies suggest that FLINT metabolite binds FasL with an apparent lower affinity than FLINT. Therefore, the pharmaceutical utility of FLINT could be enhanced by an analog that is resistant to proteolysis at or near the 218 position. The invention disclosed herein provides such analogs.
In one embodiment, the invention relates to a FLINT analog that is resistant to proteolysis between positions 218 and 219 of SEQ ID NO:1, and/or between positions 247 and 248 of SEQ ID NO:3 in vivo and/or in vitro.
In another embodiment, the invention relates to a FLINT analog that is substantially resistant to proteolysis between positions 218 and 219 of SEQ ID NO:1, and/or between positions 247 and 248 of SEQ ID NO:3 in vivo and/or in vitro.
In another embodiment, the invention relates to a FLINT C) analog that is resistant to proteolysis by a trypsin-like protease between positions 218 and 219 of SEQ ID NO:1, and/or between positions 247 and 248 of SEQ ID NO: 3 in vivo and/or in vitro.
In another embodiment, the invention relates to a FLINT analog that is resistant to proteolysis by a serine protease, for example, trypsin, thrombin, or chymotrypsin between positions 218 and 219 of SEQ ID NO:1, and/or between positions 247 and 248 of SEQ ID NO:3, in vivo and/or in vitro.
In another embodiment, the invention relates to a FLINT analog that is resistant to proteolysis by a trypsin-like protease between positions 218 and 219 of SEQ ID NO:1, and/or between positions 247 and 248 of SEQ ID NO:3, said analog comprising a polypeptide that is at least about 80% identical; alternatively at least about 90% identical; alternatively at least about 95% identical; alternatively at least 96% identical; alternatively at least 97% identical; alternatively at least 98% identical; alternatively still, at least 99% identical with SEQ ID NO:1 and/or SEQ ID NO:3.
In another embodiment, the invention relates to a FLINT analog that is resistant to proteolysis by a trypsin-like protease between positions 218 and 219 of SEQ ID NO:1, and/or between positions 247 and 248 of SEQ ID NO:3, said analog comprising a polypeptide that is at least about 10% identical; alternatively at least 20% identical; alternatively at least 30% identical; alternatively at least 40% identical; alternatively at least 50% identical; alternatively at least 60% identical; alternatively at least 70% identical, alternatively at least 80% identical, alternatively still, at least 90% identical with residues 214 through 222 of SEQ ID NO:1 and/or residues 243 through 251 of SEQ ID NO:3.
In another embodiment, the invention relates to a FLINT analog comprising one or more amino acid substitutions) deletion(s), or addition(s) in the region comprising amino acids 214-222 of SEQ ID NO:1 and/or amino acids 243-251 of SEQ ID NO:3.
In another embodiment, the invention relates to a FLINT analog comprising one or more amino acid substitutions), deletions, or addition(s) in the region comprising amino acids 215-218 of SEQ ID NO:1 and/or amino acids 243-251 of SEQ ID NO:3.
In another embodiment, the invention relates to a FLINT analog comprising one or more amino acid substitution(s) in the region 214-222 of SEQ ID NO:1, and/or amino acids 243-251 of SEQ ID NO:3.
In another embodiment, the invention relates to a FLINT analog comprising an amino acid substitution (s) in the region comprising amino acids 214-222 of SEQ ID NO:1, selected from the group consisting of:
a. Pro at position 215 is replaced by any naturally occurring amino acid other than Pro;
b. Thr at position 216 is replaced by any naturally occurring amino acid other than Thr;
c. Pro at position 217 is replaced by any naturally occurring amino acid other than Pro;
d. Arg at position 218 is replaced by any naturally occurring amino acid other than Arg;
e. Ala at position 219 is replaced by any naturally occurring amino acid other than Ala;
f. Gly at position 220 is replaced by any naturally occurring amino acid other than Gly;
g. Arg at position 221 is replaced by any naturally occurring amino acid other than Arg;
h. Ala at position 222 is replaced by any naturally occurring amino acid other than Ala.
In another embodiment, the invention relates to a FLINT analog comprising an amino acid substitution in the region comprising amino acids 214-222 of SEQ ID NO:1, selected from the group consisting of:
a. Gly at position 214 is replaced by a positively charged amino acid that is not Gly;
b. Pro at position 215 is replaced by a positively charged amino acid that is not Pro;
c. Thr at position 216 is replaced by a positively charged amino acid that is not Thr;
d. Pro at position 217 is replaced by a positively charged amino acid that is not Pro;
e. Arg at position 218 is replaced by a positively charged amino acid that is not Arg;
f. Ala at position 219 is replaced by a positively charged amino acid that is not Ala;
g. Gly at position 220 is replaced by a positively charged amino acid that is not Gly;
h. Arg at position 221 is replaced by a positively charged amino acid that is not Arg;
i. Ala at position 222 is replaced by a positively charged amino acid that is not Ala.
In another embodiment, the invention relates to a FLINT analog comprising an amino acid substitution in the region comprising amino acids 214-222 of SEQ ID NO:1, selected from the group consisting of:
a. Gly at position 214 is replaced by a negatively charged amino acid that is not Gly;
b. Pro at position 215 is replaced by a negatively charged amino acid that is not Pro;
c. Thr at position 216 is replaced by a negatively charged amino acid that is not Thr;
d. Pro at position 217 is replaced by a negatively charged amino acid that is not Pro;
e. Arg at position 218 is replaced by a negatively charged amino acid that is not Arg;
f. Ala at position 219 is replaced by a negatively charged amino acid that is not Ala;
g. Gly at position 220 is replaced by a negatively charged amino acid that is not Gly;
h. Arg at position 221 is replaced by a negatively charged amino acid that is not Arg;
i. Ala at position 222 is replaced by a negatively charged amino acid that is not Ala.
In another embodiment, the invention relates to a FLINT analog comprising an amino acid substitution in the region comprising amino acids 214-222 of SEQ ID NO:1, selected from the group consisting of:
a. Gly at position 214 is replaced by a polar uncharged amino acid that is not Gly;
b. Pro at position 215 is replaced by a polar uncharged amino acid that is not Pro;
c. Thr at position 216 is replaced by a polar uncharged amino acid that is not Thr;
d. Pro at position 217 is replaced by a polar uncharged amino acid that is not Pro;
e. Arg at position 218 is replaced by a polar uncharged amino acid that is not Arg;
f. Ala at position 219 is replaced by a polar uncharged amino acid that is not Ala;
g. Gly at position 220 is replaced by a polar uncharged amino acid that is not Gly;
h. Arg at position 221 is replaced by a polar uncharged amino acid that is not Arg;
i. Ala at position 222 is replaced by a polar uncharged amino acid that is not Ala.
In another embodiment, the invention relates to a FLINT analog comprising an amino acid substitution in the region comprising amino acids 214-222 of SEQ ID NO:1, selected from the group consisting of:
a. Gly at position 214 is replaced by a nonpolar amino acid that is not Gly;
b. Pro at position 215 is replaced by a nonpolar amino acid that is not Pro;
c. Thr at position 216 is replaced by a nonpolar amino acid that is not Thr;
d. Pro at position 217 is replaced by a nonpolar amino acid that is not Pro;
e. Arg at position 218 is replaced by a nonpolar amino acid that is not Arg;
f. Ala at position 219 is replaced by a nonpolar amino acid that is not Ala;
g. Gly at position 220 is replaced by a nonpolar amino acid that is not Gly;
h. Arg at position 221 is replaced by a nonpolar amino acid that is not Arg;
i. Ala at position 222 is replaced by a nonpolar amino acid that is not Ala.
In another embodiment, the invention relates to a FLINT analog comprising an amino acid substitution in the region comprising amino acids 214-222 of SEQ ID NO:1, selected from the group consisting of:
a. Arg at position 218 is replaced by Gln;
b. Arg at position 218 is replaced by Glu;
c. Thr at position 216 is replaced by Pro;
d. Arg at position 218 is replaced by Ala;
e. Arg at position 218 is replaced by Gly;
f. Arg at position 218 is replaced by Ser;
g. Arg at position 218 is replaced by Val
h. Arg at position 218 is replaced by Tyr;
i. Pro at position 217 is replaced by Tyr
j. Thr at position 216 is replaced by Pro, and Arg at position 218 is replaced by Gln.
In another embodiment, the present invention relates to a FLINT analog comprising SEQ ID NO:1 wherein Arg at position 34 is replaced by Asn, Asp at position 36 is replaced by Thr, and Arg at position 218 is replaced by Gln, Glu, Ala, Gly, Ser. Val, or Tyr.
In another embodiment, the present invention relates to a FLINT analog comprising SEQ ID NO:1 wherein Arg at position 34 is replaced by Asn, Asp at position 36 is replaced by Thr, Asp at position 194 is replaced by Asn, Ser at position 196 is replaced by Thr, and Arg at position 218 is replaced by Gln, Glu, Ala, Gly, Ser, Val, or Tyr.
In another embodiment, the present invention relates to a FLINT analog comprising one or more amino acid substitution(s) within SEQ ID NO:1 wherein Arg at position 34 is replaced by Asn, Asp at position 36 is replaced by Thr, and Arg at position 218 is replaced by an amino acid selected from the group consisting of:
a. any naturally occurring amino acid that is not Arg;
b. any positively charged amino acid that is not Arg;
c. any negatively charged amino acid that is not Arg;
d. any polar uncharged amino acid that is not Arg;
e. any nonpolar amino acid that is not Arg; and
f. an amino acid that is Glu, Gln, Ala, Gly, Ser, Val, or Tyr.
In another embodiment, the present invention relates to a FLINT analog comprising one or more amino acid substitutions within SEQ ID NO:1 wherein Arg at position 34 is replaced by Asn, Asp at position 36 is replaced by Thr, Asp at position 194 is replaced by Asn, Ser at position 196 is replaced by Thr, and Arg at position 218 is replaced by an amino acid selected from the group consisting of:
a. any naturally occurring amino acid that is not Arg;
b. any positively charged amino acid that is not Arg;
c. any negatively charged amino acid that is not Arg;
d. any polar uncharged amino acid that is not Arg;
e. any nonpolar amino acid that is not Arg; and
f. an amino acid that is Glu, Gln, Ala, Gly, Ser, Val, or Tyr.
In another embodiment, the present invention relates to a FLINT analog comprising one or more amino acid substitution(s) within SEQ ID NO:1 wherein Ser at position 132 is replaced by Asn, and Arg at position 218 is replaced by an amino acid selected from the group consisting of:
a. any naturally occurring amino acid that is not Arg;
b. any positively charged amino acid that is not Arg;
c. any negatively charged amino acid that is not Arg;
d. any polar uncharged amino acid that is not Arg;
e. any nonpolar amino acid that is not Arg; and
f. an amino acid that is Glu, Gln, Ala, Gly, Ser, Val, or Tyr.
Another embodiment relates to a nucleic acid encoding a protease-resistant FLINT analog of the present invention.
In another embodiment, the invention relates to a protease resistant FLINT analog that is encoded by a nucleic acid that hybridizes to SEQ ID NO:2 under high stringency conditions.
In another embodiment, the present invention relates to a nucleic acid that encodes a protease resistant FLINT analog, said nucleic acid hybridizing to SEQ ID NO:2 under high stringency conditions.
In another embodiment, the present invention relates to a vector comprising a nucleic acid encoding a protease-resistant FLINT analog.
In another embodiment the invention relates to therapeutic and clinical uses of a protease resistant FLINT analog to prevent or treat a disease or condition in a mammal in need of such prevention or treatment.
In another embodiment the invention relates to therapeutic and clinical uses of a protease resistant FLINT analog to prevent or treat acute lung injury (ALI), acute respiratory distress syndrome (ARDS), ulcerative colitis, and to facilitate organ preservation for transplantation.
In another embodiment the present invention relates to a pharmaceutical composition comprising a protease resistant FLINT analog.
In another embodiment the present invention relates to the use of FLINT analog to inhibit T lymphocyte activation.
In another embodiment, the present invention relates to the use of FLINT analog to prevent or treat chronic obstructive pulmonary disease (COPD).
In another embodiment, the present invention relates to the use of FLINT analog to prevent or treat pulmonary fibrosis (PF).
In another embodiment, the present invention relates to a method for producing a protease resistant FLINT analog, said analog being resistant to proteolysis by a trypsin-like protease between positions 218 and 219 of SEQ ID NO:1 (alternatively between positions 247 and 248 of SEQ ID NO:3), comprising the step of altering the amino acid sequence in the region at and/or between positions 214 to 222 of SEQ ID NO:1, as described herein.